Primary hyperoxaluria is a rare, genetic disorder that results from an excessive endogenous synthesis of oxalate. Glyoxylate reductase (GR) is a key enzyme in this biosynthetic pathway, converting glyoxylate, the main precursor of oxalate, to glycolate and thereby limiting its conversion to oxalate. The long term goals of this project are to develop an in depth understanding of the pathways associated with GR activity and glyoxylate and oxalate synthesis, to determine the metabolic consequences of an absence of GR activity, and to identify therapeutic strategies that limit oxalate synthesis when the activity is absent. To assist with these goals we have developed a cell line with reduced GR activity and have obtained a knockout (KO) species of mouse lacking GR activity. This proposal has three specific aims. The first aim is to determine the phenotype of the KO mouse, measuring anion concentrations in blood, urine and tissues using ion chromatography coupled to mass detection (IC/MS). The second aim is designed to test the hypothesis that inhibiting hydroxyproline oxidase (HPOX) will be an effective therapeutic strategy for decreasing the increased oxalate synthesis associated with GR deficiency. To establish proof of principle, RNA interference will be utilized to inhibit HPOX activity and decrease oxalate synthesis. The KO mouse will be used as a model for these studies. In the third specific aim, the functional role of GR in red blood cells will be examined. These cells have a simplified metabolism in comparison to other cells and preliminary data indicate that these cells contain glycolate, glyoxylate and oxalate, as well as GR activity. It is hypothesized that GR functions to limit oxalate production and to synthesize glycolate, a potential substrate for phosphoglycolate synthesis. These hypotheses will be tested in cells from KO mice and human cells incubated with glycolate and glyoxylate. These studies will increase our understanding of the metabolism associated with oxalate synthesis and will potentially identify novel therapeutic strategies to decrease oxalate synthesis in primary hyperoxaluria.